By “detecting” is meant qualitatively analysing for the presence or absence of a substance. By “determining” is meant quantitatively analysing for the amount of a substance.
Ractopamine is a phenethanolamine leanness-enhancing agent recently approved as a feed additive for swine in the United States. Hogs administered dietary ractopamine show increased growth rates and feed efficiencies and decreased fat deposition, relative to untreated animals.
Phenethanolamine β-agonists have a history of being used for off-label purposes by livestock producers hoping to improve the economics of livestock production. Improper use of β-agonists can cause a serious risk to human health due to the residues they leave in the meat and other foodstuffs of animal origin. Indeed, in Europe, all β-agonists are banned for use in livestock and for improving athletic performance under EU Directive 96/22/EC.
The presence of drug residues in animal tissues is a concern for food safety, especially when the compound has been used illegally or in a manner proscribed by regulatory officials (off-label use). In an effort to combat such illicit use of β-agonist compounds, regulatory organisations around the world test animal tissues and body fluids for the presence of such illicit drugs.
Specific binding actions, such as antibody-antigen interactions, have been used extensively in immunoassays to detect a variety of substances present in biological fluids. Thus, for example, radioimmunoassays could be used for the determination of phenethanolamines such as ractopamine, isoxsuprine and ritodrine. Radioimmunoassays are very sensitive but do require radionucleotide tracers. Enzyme-linked immunosorbent assays (ELISAs) are a non-radioactive alternative which could be used for the qualitative and quantitative determination of phenethanolamines such as, but not limited to, ractopamine, isoxsuprine and ritodrine.
Haasnoot et al (The Analyst (1994), 119, “Determination of fenoterol and ractopamine in urine by enzyme immunoassay”, 2675–2680) disclose a urinary enzyme immunoassay for ractopamine. The Haasnoot antibody was derived from a fenoterol derivative and showed 20% cross-reactivity with ractopamine, but the analysis of ractopamine did not correlate well with GC-MS analysis. Elliott et al (The Analyst (1998), 123, “Screening and confirmatory determination of ractopamine residues in calves treated with growth promoting doses of the β-agonist”, 1103–1107) disclose an immunoassay for ractopamine residues. The Elliott antibody, when used in an immunoassay where the samples were enzymatically hydrolysed prior to analysis, correlated well with LC-MS-MS but, as with Haasnoot et al, the antibodies were raised in a non-targeted fashion, against a fentoterol derivative.
Shelver and Smith (Journal of Immunoassay (2000), 21(1), “Development of an immunoassay for the β-adrenergic agonist ractopamine”, 1–23) disclose preparation of polyclonal antibodies generated from ractopamine-hemiglutarate-KLH. The hapten is prepared by reaction of ractopamine with glutaric anhydride, followed by conjugation with an antigenicity-conferring carrier material. The method employed to synthesize ractopamine-hemiglutarate is uncontrolled and will result in derivatisation of ractopamine at any one of positions 1, 10, 10′ and N. The Shelver and Smith antibody generated shows a sensitivity (IC50) of 4.2 ng/ml toward ractopamine. The antibody also exhibits 33% cross-reactivity with dobutamine.
Shelver et al (J. Agric. Food Chem. 48 (2000), “Production and characterisation of a monoclonal antibody against the β-adrenergic agonist ractopamine”, 4020–4026 and their U.S. Pat. No. 6,274,334) disclose the generation of a monoclonal antibody, in which ractopamine-glutarate-keyhole limpet haemocyanin was used as the antigen for antibody generation. The antibody clone selected (5G10) shows 5.3% cross-reactivity with dobutamine and 3.6% cross-reactivity with ritodrine.
A polyclonal antibody is generated by repeated immunisation of a mammalian host with the target immunogen. The resulting antiserum is harvested and the antibodies isolated. To generate a monoclonal antibody, lymphocytes from an immunized animal are fused with a myeloma cell line to produce hybrid cells or hybridomas. These hybridomas can be cloned for production of the secreted monoclonal antibody. A monoclonal antibody will recognize a single epitope, whereas a polyclonal antiserum may recognize several epitbpes on the same antigen. Hence, monoclonal antibody technology can usually be applied to generate an antibody with greater specificity for the target immunogen.
In the aforementioned Shelver et al, a ‘scattergun’ approach has been used to generate polyclonal antibodies to ractopamine. The uncontrolled method used to prepare the ractopamine hapten suggests that the resulting immunogen is composed of a mixture of ractopamine linked to keyhole limpet haemocyanin (KLH) at any one of positions 1, 10, 10′ and N. Immunisation with such an immunogen will result in the generation of antisera with a diversity of specificity. This is borne out in the cross-reactivity data presented in the Shelver reference. Their polyclonal antibody cross-reacts 33% with dobutamine, while their monoclonal antibody cross-reacts 5.3% with dobutamine. In contrast, the targeted introduction of a crosslinker group onto a single phenolic hydroxyl group of the target hapten, according to the present invention, produces a surprising effect (a heterofunctional crosslinker as defined herein, is a structure incorporating one or more functional groups containing one or more hetero-atoms, that links, for example through covalent bonding, with a substrate (in this case a hapten), the crosslinker also being able to link to a peptide, polypeptide or protein or a detectable labelling agent). Antibodies generated to the present haptens are highly specific and do not cross-react significantly with dobutamine. In the case of ractopamine, the antibody generated to the position 10 derivative cross-reacts 0.186% with dobutamine, while the antibody generated to the position 10′ derivative exhibits cross-reactivity of <0.8% with dobutamine. The only explanation for the cross-reactivity of the Shelver antibodies with dobutamine is that Shelver et al have generated antibodies to ractopamine derivatised at the position 1 hydroxyl (hydroxyl group on the aliphatic chain). This hydroxyl group is absent in dobutamine. The presence of a single hydroxyl group on one of the aromatic rings of the hapten derivatives used in the present application results in a high degree of specificity for ractopamine, isoxsuprine and ritodrine. The antibodies of the present application are also considerably more sensitive than the Shelver polyclonal and monoclonal antibodies. For ractopamine, the antibody to the position 10 derivative has an IC50 of 0.082 ng/ml, while the antibody to the position 10′ derivative has an IC50 of 0.202 ng/ml. The present antibodies exhibit improved specificity and sensitivity. The present application illustrates how a superior antibody can be generated using polyclonal antibody technology and the process of targeted derivatisation.
None of the prior art known to the inventors either discloses or suggests preparing phenethanolamines haptens such as, but not limited to, ractopamine, isoxsuprine or ritodrine haptens by the method disclosed herein. The present method allows for the controlled coupling of a crosslinking group to a single phenolic hydroxyl group of the hapten. The present invention, by allowing the preparation of haptens and, therefore, immunogens derivatised at a single phenolic hydroxyl group, facilitates preparation of antibodies to immunogens derivatised at a single phenolic hydroxyl group. None of the prior art discloses or suggests targeted derivatisation of phenethanolamines such as, but not limited to, ractopamine, isoxsuprine and ritodrine analogues at a single phenolic hydroxy group. Such haptens also form part of the present invention.